Insulin promotes the expression of the gluconeogenic rate-limiting enzymes phosphoenolpyruvate carboxykinase (Pepck) and glucose 6-phosphatase (G6pase) through PI3k/Akt/mTOR signaling pathway in goose hepatocytes

To identify what makes insulin have an activating or inhibiting role in gluconeogenesis in goose hepatocytes and whether insulin regulates PEPCK and G6Pase through the PI3k/Akt/mTOR pathway or not, goose primary hepatocytes were isolated and cultured in vitro. After 12h cultured in serum-free medium, hepatocytes were incubated for 24 h in the medium with no addition (control) or with the addition of 50, 100, and 150 nM of insulin, 1000 nM NVP-BEZ235, or co-addition of 150nM insulin and 1000nM NVP-BEZ235. Glucose concentration and PEPCK and G6Pase expression were determined. The results showed that PEPCK and G6Pase mRNA levels and activities were up regulated in the 50, 100, and 150nM insulin treatments, while glucose concentration was not significantly altered (p > 0.05). Compared with the activation role of 150nM insulin alone, the co-treatment with1000nM NVP-BEZ235 and 150nM insulin significantly down regulated PEPCK mRNA level and G6Pase protein activity (p < 0.05). However, there is a different result on mRNA level of G6Pase. In conclusion, G6Pase and PEPCK are up regulated by insulin through PI3k/Akt/mTOR pathway in goose hepatocytes. However, G6Pase mRNA and protein levels may be regulated by insulin through different signaling pathways.(AU)

Insulin promotes the expression of the gluconeogenic rate-limiting enzymes phosphoenolpyruvate carboxykinase (Pepck) and glucose 6-phosphatase (G6pase) through PI3k/Akt/mTOR signaling pathway in goose hepatocytes

To identify what makes insulin have an activating or inhibiting role in gluconeogenesis in goose hepatocytes and whether insulin regulates PEPCK and G6Pase through the PI3k/Akt/mTOR pathway or not, goose primary hepatocytes were isolated and cultured in vitro. After 12h cultured in serum-free medium, hepatocytes were incubated for 24 h in the medium with no addition (control) or with the addition of 50, 100, and 150 nM of insulin, 1000 nM NVP-BEZ235, or co-addition of 150nM insulin and 1000nM NVP-BEZ235. Glucose concentration and PEPCK and G6Pase expression were determined. The results showed that PEPCK and G6Pase mRNA levels and activities were up regulated in the 50, 100, and 150nM insulin treatments, while glucose concentration was not significantly altered (p > 0.05). Compared with the activation role of 150nM insulin alone, the co-treatment with1000nM NVP-BEZ235 and 150nM insulin significantly down regulated PEPCK mRNA level and G6Pase protein activity (p < 0.05). However, there is a different result on mRNA level of G6Pase. In conclusion, G6Pase and PEPCK are up regulated by insulin through PI3k/Akt/mTOR pathway in goose hepatocytes. However, G6Pase mRNA and protein levels may be regulated by insulin through different signaling pathways.

PI3K-Akt-mTOR signal inhibition affects expression of genes related to endoplasmic reticulum stress.

PI3K-Akt-mTOR signaling pathway is associated with endoplasmic reticulum (ER) stress. However, it is not clear how this signaling pathway affects the ER stress. The present study aimed to determine whether the PI3K-Akt-mTOR signaling pathway regulates tunicamycin (TM)-induced increases in mRNA levels of genes involved in the ER stress, to help elucidate the mechanism by which this pathway affects the ER stress in primary goose hepatocytes. Primary hepatocytes were isolated from geese and cultured in vitro. After 12 h in a serum-free medium, the hepatocytes were incubated for 24 h in a medium with either no addition (control) or with supplementation of TM or TM together with PI3K-Akt-mTOR signaling pathway inhibitors (LY294002, rapamycin, NVP-BEZ235). Thereafter, the expression levels of genes involved in the ER stress (BIP, EIF2a, ATF6, and XBP1) were assessed. The results indicated that the mRNA level of BIP was up-regulated in 0.2, 2, and 20 µM TM treatment group (P < 0.05), whereas the mRNA levels of EIF2a, ATF6, and XBP1 were up-regulated in the 2 µM TM treatment group (P < 0.05). However, the TM mediated induction of mRNA levels of genes involved in the ER stress (BIP, EIF2a, ATF6, and XBP1) was down-regulated after the treatment with PI3K-Akt-mTOR pathway inhibitors (LY294002, NVP-BEZ235, and rapamycin). Therefore, our results strongly suggest that the PI3K-Akt-mTOR signaling pathway might be involved in the down-regulation of the TM-induced ER stress in primary goose hepatocytes.

Molecular cloning, characterization and expression analysis of WAG-1 in the pistillody line of common wheat.

Wheat WAG-1 is a C-class MADS-box gene, which is orthologous to AGAMOUS in Arabidopsis. In this study, we report the cloning, characterization, and expression patterns of WAG-1 in the pistillody mutant HTS-1 and its sib-line CSTP. The cDNA of WAG-1 was found to be 765 bp in length, which was equal to the length of its open reading frame, encoding 254 amino acids. The location of WAG-1 revealed that it has three homologous genes from the short arm of chromosome 1A, 1B, and 1D. Their genomic sequences were determined to be 5864, 6454, and 6447 bp long, respectively, and possessed seven exons and six introns. Young spikes from HTS-1 contained higher levels of WAG-1 transcript than did those from CSTP, and the transcript levels in the young spikes (7-10 mm in length) of HTS-1 increased 3.3-fold relative to those of the CSTP line. The transcript level in the pistil and pistil-like stamens of HTS-1 was over 2-fold higher than that in the stamens of CSTP, and expression in the pistil-like stamens of HTS-1 was slightly higher than that in its pistils. These data provide a basis for future research into the function of WAG-1, and offer further insight into the molecular mechanism of the pistillody mutation in common wheat.

Identification of differentially expressed genes in a pistillody common wheat mutant using an annealing control primer system.

HTS-1 is a new kind of pistillody wheat. All or parts of its stamen are transformed into pistils or pistil-like structures, and it has more seed sets per floret than normal wheat under normal cultivation conditions. To investigate the expression divergence in this mutant, an annealing control primer system was used to identify differentially expressed genes (DEGs) in the young spikelets. As a result, three DEGs, including HDB2, HGF2, and HCG4, were detected, with variable expression in HTS-1 and the control. After further confirmation using real-time reverse transcription polymerase chain reaction analysis, these genes were overexpressed in HTS-1 wheat. NGF2 was identified in the double ridge to floret differentiation stages; HDB2 and HCG4 were identified in the stage of pistil and stamen-differentiating. Therefore, we inferred that the homeotic transformation of stamens into pistil-like structures occurred during the early stage of stamen development. Sequence alignment analysis revealed that HDB2 encodes a putative protein of 189 amino acids, with high homology to the DEAD-box ATP-dependent RNA helicase, and HCG4 was identical to the Chinese spring wheat cDNA clone predicted protein according to GenBank. However, NGF2 was not found to have significant similarity to any reported proteins, suggesting it is a new functional gene in wheat. The results suggest that HDB2, HCG4, and HGF2 are minor genes contributing to pistillody trait formation in HTS-1.

Visualization and body distribution of [¹³¹I]-herceptin in nude mice with BT-474 breast carcinoma.

The study aimed to investigate the bio-distribution and radio-immuno-imaging features of [(131)I]-herceptin in nude mice with BT-474 breast carcinoma. [(131)I]-Herceptin was administrated by tail intravenous injection to the nude mice with BT-474 breast carcinoma. Radiocounting was performed at 4, 12, 24, 48, and 96 h after administration. The activity ratio in the tumor tissue and non-tumor tissue (T/NT) and the radiocounting percentage per gram tissue to the injected dose (%ID/g) were calculated. The nude mice with BT-474 breast carcinoma were also visualized continuously by single photon emission computed tomography at 2, 4, 8, 12, 24, 48, and 96 h after the injection of [(131)I]-herceptin. Nude mice with MDA-MB-231 used as the control group were subjected to the same analyses. Clear tumor images were obtained after the injection of [(131)I]-herceptin in nude mice with BT-474 breast carcinoma. The images were the clearest at 24 h after the injection and remained clear even at 96 h. The T/NT ratio and %ID/g in the tumor tissues of nude mice with BT-474 were both significantly higher than those of the control group (P < 0.01). [(131)I]-Herceptin displays tumors clearly in the nude mice with BT-474 and accumulates well in the tumor tissues.

Cloning and characterization of thioredoxin h in the three-pistil line of common wheat.

Thioredoxin h (Trxh) is a ubiquitous protein that reduces disulfides in target proteins, and is itself reduced by NADPH-thioredoxin reductase. In the current study, the complementary DNA sequence and the genomic sequence of the three-pistil (TP) line of common wheat (Triticum aestivum L.) were obtained from spikes through reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR. Sequence alignment of amino acids of TPTrxh then allowed for predictions of its physicochemical properties, secondary structures, tertiary structures, and functional domains. Furthermore, the TPTrxh gene was overexpressed in Escherichia coli and its activity was demonstrated using a dithiothreitol-dependent insulin assay. The expression patterns of TPTrxh were analyzed through real-time RT-PCR in different tissues and across different developmental stages of young spikes. The complementary DNA of TPTrxh was found to be 411 bp in length, encoding 118 amino acids. Its genomic sequence was determined to be 2632 bp, possessing 3 exons and 2 introns. Functional domain analysis indicated that TPTrxh contained a WCGPC motif located at the end of the second ß-fold and on the initial side of the second α-helix. The TPTrxh protein reduces intramolecular and intermolecular disulfide bridges in target proteins. Young spikes contain higher levels of TPTrxh transcripts than do stems and leaves. The transcript levels in the young spikes (2-5 mm in length) of the Chinese Spring TP line increased 2.84-fold relative to those of young spikes (2-5 mm in length) of the Chinese Spring line. These data provide a basis for future research into the function of Trxh, and offer further insight into the molecular mechanism of the TP mutation in wheat.